Research On The Infrared Spectrum Detection Of DNA Damage Induced By Nitrosoureas

The nitrosourea compounds are an important class of DNA alkylating agents.The detection of DNA alkylation damages plays an important role in cancer research.The basic method for the rapid determination of DNA damages by infraredspectroscopy (IR) has been established in this thesis. The positive reagents, ethyliodide and methyl methane-sulfonate (MMS), were used which were interacted withDNA and comfirmed the feasibility of IR in the determination of DNA alkylationdamages. And then the DNA damages induced by1-methyl-1-nitrosourea (MNU) aswell as Nimustine (ACNU) were explored, respectively. The contents are mainlyinvolved in the following three parts.Firstly, a rapid, simple and convenient method with Fourier transform infrared(FTIR) spectroscopy was established for the characterization of oligo DNA and calfthymus DNA (CT-DNA). And the two ways of IR, transmission method andattenuated total reflection (ATR) method, were compared in the detection of DNA.The results showed that the transmission method could be used for detecting both theCT-DNA and oligo DNAwith high reproducibilities. The method of ATR also couldbe used for the characterisation of oligo DNA, however, the signal was too weak to beused to characterize CT-DNA with such relatively complex structure. Meanwhile, thecharacteristic infrared peaks of the two types of DNA were affirmed. Thecharacteristic absorption bands including DNA bases, namely, guanine (G), cytosine(C), adenine (A) and thymine (T), backbone PO2group and deoxyribose C-C skeletonwere cleared in the research.Secondly, the feasibility for characterization of the DNA damage was discussedwith FTIR spectroscopy. Two positive alkylating agents, ethyl iodide and MMS, wereused to interact with CT-DNA for8hours, and then got the spectrogram bytransmission method. The result showed that ethyl iodide mainly perturbed G, A andC, causing the red shift of the characteristic spectral bands. Besides, the peak intensitygradually increased as the interaction proceeded. MMS perturbed the structure ofCT-DNA, causing the B-type change to the Z-type, and the details were as follows:the IR marker band of the backbone PO2group red shifted from1228cm-1to1220cm-1; the infrared marker band of backbone C-O (1053cm-1) increased sharply. Andthat the intensity of infrared marker bands at1713cm-1(G),1610cm-1(A) and1663cm-1(T) increased, while the intensity of IR marker band of C bases (1492cm-1) decreased.Thirdly, the interactions of CT-DNA and Oligo DNA with nitrosoureas wereexplored. Discussion on the affection of DNA interacted with the two representativenitrosoureas, MNU and ACNU, at different interaction time and drug concentrationswas carried out. The result showed that MNU perturbed G and A bases of theCT-DNA, causing the characteristic spectral bands red-shifted with the increasing ofthe intensity. With the drug concentration increasing at the first8hours, the intensityvariation and shifting of the infrared tagged bands were more remarkable. However,the PO2group band exhibited no major of intensity variation or shifting. It wasindicated that MNU-CT-DNA interacting with G and A bases, but did not lead to theconformational change of DNA. For Oligo DNA, MNU perturbed the C=O, C=Ngroups of four bases (G, A, T, C), causing the characteristic spectral bands shiftedslightly. In ACNU-CT-DNA interacting, the intensity of G bases was graduallyincreasing at the first6hours, however, it was decreasing at6-8hours, indicating thatACNU perturbed the G bases in two steps, and the second step might be due to theoccurrence of DNA cross-linking induced by Nimustine. Meanwhile, ACNU causedthe B-DNA changing to the A-DNA. ACNU interacted with Oligo DNA at the C, Tand A bases, causing the marker bands of C and T bases bule shifted and the intensityincreased; the intensity of the characteristic spectral band of A bases increasedsignificantly.